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This protocols describes the generation of islet-like clusters. These islet clusters contain 2%–8% human c-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. The insulin content measured in the islets is higher than that of human fetal islets. In addition, the islet clusters contain numerous secretory granules, as determined by electron microscopy, and secrete human c-peptide in a glucose-dependent manner.